Novel Rab proteins

ABSTRACT

The present invention provides four human Rab and Rab-associated proteins (designated collectively as HRAB) and polynucleotides which identify and encode HRAB. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding HRAB and a method for producing HRAB. The invention also provides for use of HRAB and agonists, antibodies, or antagonists specifically binding HRAB, in the prevention and treatment of diseases associated with expression of HRAB. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding HRAB for the treatment of diseases associated with the expression of HRAB. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding HRAB.

[0001] This application is a continuation application of U.S.application Ser. No. 09/240,364, filed Jan. 29, 1999, which is adivisional application of U.S. application Ser. No. 08,741,411, filedOct. 29, 1996, now U.S. Pat. No. 6,124,116, issued Sep. 26, 2000, bothentitled NOVEL RAB PROTEINS, all of which applications and patents arehereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a novel human Rab and Rab-associated proteins and to the use of thesesequences in the diagnosis, prevention, and treatment of choroideremia,AIDS, and cancer.

BACKGROUND OF THE INVENTION

[0003] Transport of material between the different subcellularcompartments of eukaryote cells often requires carrier vesicles, whichbud from a donor organelle and fuse with the recipient one. Rab proteinsare low molecular weight guanidine triphosphatases (GTPases) of the Rassuperfamily which are localized to the membrane surfaces of organelles.They appear to be involved in the regulation of intracellular vesiculartransport in both exocytic and endocytic pathways. They may also beinvolved in the complex and critical processes of organellefragmentation and restructuring that occur each cell cycle. Rab proteinscycle between active GTP-bound and inactive GDP-bound conformations.

[0004] Newly formed Rab proteins associate with Rab escort proteins(REPs) in the cell cytosol. Rab proteins are then stably isoprenylatedby the covalent addition of two 20-carbon geranylgeranyl groups tocarboxy-terminal cysteine residues (Khosravi-Far R et al (1991) ProcNatl Acad Sci 88: 6264-6268). Prenylation occurs by Rab geranylgeranyltransferase (GGTase) and is essential for Rab protein function andmembrane localization. A deficiency in prenylation of one particular Rableads to choroideremia, a form of retinal degeneration that may causeblindness (Seabra MC et al (1996) J Biol Chem 270: 24420-24427; Seabraet al (1993) Science 259: 377-381). Each of the more than 30 Rabproteins identified appears to have characteristic intracellulardistribution and may function in distinct transport events. REPs helptransfer newly prenylated Rab proteins to the appropriate organellemembrane.

[0005] The amino acid sequence of Rab proteins reveal conservedGTP-binding domains that are characteristic among Ras superfamilymembers (Zahraoui A et al (1989) J Biol Chem 264: 12394-123401). GTPbinding or conversion from GDP to GTP form occurs en route to theorganelle membrane. Experimental evidence shows that GTP-bound Rabproteins are directed into nascent transport vesicles where theyinteract with SNARE factors, a complex of proteins that direct vesicletargeting and fusion. Following vesicle transport, GTPase activatingproteins (GAPs) in the target membrane convert Rab proteins to theGDP-bound form. A cytosolic protein, guanine-nucleotide dissociationinhibitor (GDI) helps return GDP-bound Rab proteins to their membrane oforigin.

[0006] Rab proteins appear to play a role in mediating the function of aviral gene, Rev, which is essential for replication of HIV-1, the virusresponsible for AIDS (Flavell RA et al (1996) Proc Natl Acad Sci 93:4421-4424). Rab proteins, when overexpressed, can significantly enhanceRev function. Furthermore, mutational analysis suggests that Rev proteinhas a nuclear signal domain that is necessary for localization into thecell nucleus and is likely to be a Rab protein binding site (Flavell etal, supra).

[0007] Both the inhibition of vesicle transport and organellefragmentation during mitosis are due to an inhibition of vesicle fusion,which occurs while vesicle budding continues. Protein phosphorylation byCdc2 protein kinase is a key regulatory event in mitosis. Toumikoski Tet al has shown that addition of Cdc2 protein kinase to interphase cellextracts inhibits vesicle fusion (1989, Nature 342: 942-945).Furthermore, low GTP-gamma-S concentrations, which are likely to blockRab protein GTPase activity, inhibit the fusion reaction, suggestingthat Rab proteins could be mediating this critical cell cycle event.Loss of cell cycle control is a key characteristic of all human cancers.

[0008] The discovery of additional Rab and Rab-associated genes and theproteins encoded provides potential agents which are more effective thancurrently available therapeutic agents in the diagnosis and treatment ofchoroideremia, AIDS, and cancer. Thus, the new Rab and Rab-associatedproteins would satisfy a need in the art by providing new means for thediagnosis, prevention, or treatment of choroideremia, AIDS, and cancer.

SUMMARY OF THE INVENTION

[0009] The present invention features novel Rab and Rab-associatedproteins hereinafter designated individually as HRABA, HRABB, HRABC,HRABD, and collectively as HRAB and characterized as having homology toRab and Rab-associated proteins and intracellular transport activity.

[0010] Accordingly, the invention features substantially purified Raband Rab-associated proteins HRABA, HRABB, HRABC, and HRABD, havingintracellular transport activity and as shown in amino acid sequences ofSEQ ID NOS: 1, 3, 5, and 7, respectively.

[0011] One aspect of the invention features isolated and substantiallypurified polynucleotides that encode HRABA, HRABB, HRABC, and HRABD. Ina particular aspect, the polynucleotides are the nucleotide sequences ofSEQ ID NOS: 2, 4, 6, and 8, respectively.

[0012] The invention also relates to a polynucleotide sequencescomprising the complement of SEQ ID NOS: 2, 4, 6, and 8 or variantsthereof. In addition, the invention features polynucleotide sequenceswhich hybridize under stringent conditions to SEQ ID NOS: 2, 4, 6, and8.

[0013] The invention additionally features nucleic acid sequencesencoding polypeptides, oligonucleotides, peptide nucleic acids (PNA),fragments, portions or antisense molecules thereof, and expressionvectors and host cells comprising polynucleotides that encode HRAB. Thepresent invention also features antibodies which bind specifically toHRAB, and pharmaceutical compositions comprising substantially purifiedHRAB. The invention also features the use of agonists and antagonists ofHRAB.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of HRABA. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.,Ltd, San Bruno, Calif.).

[0015]FIGS. 2A, 2B, and 2C show the amino acid sequence (SEQ ID NO:3)and nucleic acid sequence (SEQ ID NO:4) of HRABB.

[0016]FIGS. 3A, 3B, and 3C show the amino acid sequence (SEQ ID NO:5)and nucleic acid sequence (SEQ ID NO:6) of HRABC.

[0017]FIGS. 4A and 4B show the amino acid sequence (SEQ ID NO:7) andnucleic acid sequence (SEQ ID NO:8) of HRABD.

[0018]FIGS. 5A and 5B show the amino acid sequence alignments amongHRABA (SEQ ID NO:1), canine rab22 (GI 437987; SEQ ID NO:9), HRABB (SEQID NO:3), rat Rab-related GTP-binding protein (GI 206543; SEQ ID NO:10),HRABC (SEQ ID NO:5), and mouse Rab17 (GI 297157; SEQ ID NO:11). Thealignment was produced using the multisequence alignment program ofDNASTAR software (DNASTAR Inc., Madison Wis.).

[0019]FIG. 6 shows the amino acid sequence alignments between HRABD (SEQID NO:7) and mouse Rab6/Rab5-associated protein (GI 722667; SEQ IDNO:12).

[0020]FIG. 7 shows the hydrophobicity plot (generated using MACDNASISPRO software) for HRABA (SEQ ID NO:1). The positive X axis reflectsamino acid position, and the negative Y axis reflects hydrophobicity.Similarly, FIG. 8 shows the hydrophobicity plot for HRABB (SEQ ID NO:3),FIG. 9 shows the hydrophobicity plot for HRABC (SEQ ID NO:5), FIG. 10shows the hydrophobicity plot for canine rab22 (SEQ ID NO:9), FIG. 11shows the hydrophobicity plot for HRABD (SEQ ID NO:7), and FIG. 12 showsthe hydrophobicity plot for mouse Rab6/Rab5-associated protein (SEQ IDNO:12).

DESCRIPTION OF THE INVENTION

[0021] Before the present protein, nucleotide sequence, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0022] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0023] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0024] Definitions

[0025] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand. Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence and fragments orportions thereof, of a naturally occurring or synthetic molecule.

[0026] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0027] “Peptide nucleic acid”, as used herein, refers to a moleculewhich comprises an oligomer to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen et al. (1993)Anticancer Drug Des. 8:53-63).

[0028] HRAB, as used herein, refers to the amino acid sequences ofsubstantially purified HRAB obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0029] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, or which has beenextended using XL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5′ and/orthe 3′ direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GCGfragment assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

[0030] A “variant” of HRAB, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0031] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0032] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0033] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0034] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic HRAB, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0035] The term “agonist”, as used herein, refers to a molecule which,when bound to HRAB, causes a change in HRAB which modulates the activityof HRAB. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to HRAB.

[0036] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to HRAB, blocks the biological orimmunological activity of HRAB. Antagonists and inhibitors may includeproteins, nucleic acids, carbohydrates, or any other molecules whichbind to HRAB.

[0037] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of HRAB. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional orimmunological properties of HRAB.

[0038] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of HRABor portions thereof and, as such, is able to effect some or all of theactions of Rab or Rab-associated protein-like molecules.

[0039] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding HRAB or the encoded HRAB.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0040] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0041] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0042] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0043] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0044] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, for thesequence “A-G-T” binds to the complementary sequence “T-C-A”.Complementarity between two single-stranded molecules may be “partial”,in which only some of the nucleic acids bind, or it may be complete whentotal complementarity exists between the single stranded molecules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands.

[0045] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0046] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0047] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences.

[0048] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0049] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1, 3, 5, or 7” encompasses the full-length human HRAB and fragmentsthereof.

[0050] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but are not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0051] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0052] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0053] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding HRABor fragments thereof may comprise a cell, chromosomes isolated from acell (e.g., a spread of metaphase chromosomes), genomic DNA (in solutionor bound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0054] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is complementary to SEQ ID NO:2, 4, 6, or 8 by northernanalysis is indicative of the presence of mRNA encoding HRAB in a sampleand thereby correlates with expression of the transcript from thepolynucleotide encoding the protein.

[0055] “Alterations” in the polynucleotide of SEQ ID NO:2, 4, 6, or 8,as used herein, comprise any alteration in the sequence ofpolynucleotides encoding HRAB including deletions, insertions, and pointmutations that may be detected using hybridization assays. Includedwithin this definition is the detection of alterations to the genomicDNA sequence which encodes HRAB (e.g., by alterations in the pattern ofrestriction fragment length polymorphisms capable of hybridizing to SEQID NO:2, 4, 6, or 8), the inability of a selected fragment of SEQ IDNO:2, 4, 6, or 8 to hybridize to a sample of genomic DNA (e.g., usingallele-specific oligonucleotide probes), and improper or unexpectedhybridization, such as hybridization to a locus other than the normalchromosomal locus for the polynucleotide sequence encoding HRAB (e.g.,using fluorescent in situ hybridization [FISH] to metaphase chromosomesspreads).

[0056] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fab, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind HRABpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromtranslated cDNA or synthesized chemically, and can be conjugated to acarrier protein, if desired. Commonly used carriers that are chemicallycoupled to peptides include bovine serum albumin and thyroglobulin. Thecoupled peptide is then used to immunize the animal (e.g., a mouse, arat, or a rabbit).

[0057] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0058] The Invention

[0059] The invention is based on the discovery of Rab and Rab-associatedproteins HRAB, the polynucleotides encoding HRAB, and the use of thesecompositions for the diagnosis, prevention, or treatment ofchoroideremia, AIDS, and cancer.

[0060] Nucleic acids encoding the human HRABA of the present inventionwere first identified in cDNA, Incyte Clone 334674 from the eosinophilcDNA library EOSIHET02 through a computer-generated search for aminoacid sequence alignments. A consensus sequence, SEQ ID NO:2, was derivedfrom the following overlapping and/or extended nucleic acid sequences(derived from the stated cDNA library): Incyte Clones 334674(EOSIHET02), 125981 (LUNGNOT01), 1370809 (BSTMNON02), and 1420225(KIDNNOT09).

[0061] In one embodiment, the invention encompasses a Rab protein(HRABA), a polypeptide comprising the amino acid sequence of SEQ IDNO:1, as shown in FIGS. 1A, 1B, and 1C. HRABA is 184 amino acids inlength. HRABA has chemical and structural homology with canine rab22 (GI437987; SEQ ID NO:9), rat Rab-related GTP-binding protein (GI 206543;SEQ ID NO:10), and mouse Rab17 (GI 297157; SEQ ID NO:11; FIGS. 5A and5B). In particular, HRABA and canine rab22 share 68% identity. Asillustrated by FIGS. 7 and 10, HRABA and canine rab22 have similarhydrophobicity which suggests that they have a similar structure. Thehomology includes conserved GTP/GDP binding domains and carboxy-terminalcysteine residues that are suitable substrates for prenylation (FIGS. 5Aand 5B). HRABA has two potential N-glycosylation sites.

[0062] Nucleic acids encoding the human HRABB of the present inventionwere first identified in cDNA, Incyte Clone 367194 from the synovialtissue cDNA library SYNORAT01 through a computer-generated search foramino acid sequence alignments. A consensus sequence, SEQ ID NO:4, wasderived from the following overlapping and/or extended nucleic acidsequences (derived from the stated cDNA library): Incyte Clones 367194(SYNORAT01), 874099 (LUNGAST01), 1214370 (BRSTTUT01), 1320737(BLADNOT04), 1452285 (PENITUT01), and 1489006 (UCMCL5T01).

[0063] In one embodiment, the invention encompasses a Rab protein(HRABB), a polypeptide comprising the amino acid sequence of SEQ IDNO:3, as shown in FIGS. 2A, 2B, and 2C. HRABB is 209 amino acids inlength. HRABB has chemical and structural homology with canine rab22 (GI437987; SEQ ID NO:9), rat Rab-related GTP-binding protein (GI 206543;SEQ ID NO:10), and mouse Rab17 (GI 297157; SEQ ID NO:11; FIGS. 5A and5B). In particular, HRABB and canine rab22 share 70% identity. Asillustrated by FIGS. 8 and 10, HRABB and canine rab22 have similarhydrophobicity which suggests that they have a similar structure. Thehomology includes conserved GTP/GDP binding domains and carboxy-terminalcysteine residues that are suitable substrates for prenylation (FIGS. 5Aand 5B). HRABB has three potential N-glycosylation sites.

[0064] Nucleic acids encoding the human HRABC of the present inventionwere first identified in cDNA, Incyte Clone 1272054 from the testiculartumor cDNA library TESTTUT02 through a computer-generated search foramino acid sequence alignments. A consensus sequence, SEQ ID NO:6, wasderived from the following overlapping and/or extended nucleic acidsequences (derived from the stated cDNA library): Incyte Clones 1272054(TESTTUT02) and 601225 (BRSTNOT02).

[0065] In one embodiment, the invention encompasses a Rab protein(HRABC), a polypeptide comprising the amino acid sequence of SEQ IDNO:5, as shown in FIGS. 3A, 3B, and 3C. HRABC is 190 amino acids inlength. HRABC has chemical and structural homology with canine rab22 (GI437987; SEQ ID NO:9), rat Rab-related GTP-binding protein (GI 206543;SEQ ID NO:10), and mouse Rab17 (GI 297157; SEQ ID NO:11; FIGS. 5A and5B). In particular, HRABC and canine rab22 share 75% identity. Asillustrated by FIGS. 9 and 10, HRABC and canine rab22 have similarhydrophobicity which suggests that they have a similar structure. Thehomology includes conserved GTP/GDP binding domains and carboxy-terminalcysteine residues that are suitable substrates for prenylation (FIGS. 5Aand 5B). HRABC has two potential N-glycosylation sites.

[0066] Nucleic acids encoding the human HRABD of the present inventionwere first identified in cDNA, Incyte Clone 358844 from the synovialtissue cDNA library SYNORAB01 through a computer-generated search foramino acid sequence alignments. A consensus sequence, SEQ ID NO:8, wasderived from the following overlapping and/or extended nucleic acidsequences (derived from the stated cDNA library): Incyte Clones 358844(SYNORAB01), 235332 (SINTNOT02), 761131 (BRAITUT02), and 966344(BRSTNOT05).

[0067] In one embodiment, the invention encompasses a Rab protein(HRABD), a polypeptide comprising the amino acid sequence of SEQ IDNO:7, as shown in FIGS. 4A and 4B. HRABD is 184 amino acids in length.HRABD has chemical and structural homology with mouseRab6/Rab5-associated protein (GI 722667; SEQ ID NO:12). In particular,HRABD and Rab6/Rab5-associated protein share 77% identity. Asillustrated by FIGS. 11 and 12, HRABD and Rab6/Rab5-associated proteinhave similar hydrophobicity which suggests that they have a similarstructure.

[0068] The invention also encompasses HRAB variants. A preferred HRABvariant is one having at least 80%, and more preferably 90%, amino acidsequence similarity to the HRAB amino acid sequence (SEQ ID NOS:1, 3, 5,or:7). A most preferred HRAB variant is one having at least 95% aminoacid sequence similarity to SEQ ID NOS:1, 3, 5, or 7.

[0069] The invention also encompasses polynucleotides which encode HRAB.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of HRAB can be used to generate recombinant molecules whichexpress HRAB. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid of SEQ ID NOS:2, 4, 6, or 8,as shown in FIGS. 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B, 3C, 4A, and 4B.

[0070] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding HRAB, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring HRAB, and all such variations are to beconsidered as being specifically disclosed.

[0071] Although nucleotide sequences which encode HRAB and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HRAB under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HRAB or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic expression host in accordance with the frequency with whichparticular codons are utilized by the host. Other reasons forsubstantially altering the nucleotide sequence encoding HRAB and itsderivatives without altering the encoded amino acid sequences includethe production of RNA transcripts having more desirable properties, suchas a greater half-life, than transcripts produced from the naturallyoccurring sequence.

[0072] The invention also encompasses production of a DNA sequence, orportions thereof, which encode HRAB and its derivatives, entirely bysynthetic chemistry. After production, the synthetic gene may beinserted into any of the many available DNA vectors and cell systemsusing reagents that are well known in the art at the time of the filingof this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding HRAB or any portionthereof.

[0073] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NOS:2, 4, 6, or 8, under variousconditions of stringency. Hybridization conditions are based on themelting temperature (Tm) of the nucleic acid binding complex or probe,as taught in Berger and Kimmel (1987; Methods Enzymol. Vol. 152), andmay be used at a defined stringency.

[0074] Altered nucleic acid sequences encoding HRAB which areencompassed by the invention include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent HRAB. The encodedprotein may also contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent HRAB. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the biological activity of HRAB is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; phenylalanine and tyrosine.

[0075] Also included within the scope of the present invention arealleles of the genes encoding HRAB. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0076] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE amplification systemmarketed by Gibco BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the MICROLAB 2200 (Hamilton, Reno,Nev.), Peltier thermal cycler (PTC200; MJ Research, Watertown, Mass.),and the ABI 377 DNA sequencers (Perkin Elmer).

[0077] The polynucleotide sequences encoding HRAB may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Gobinda et al. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0078] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0079] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

[0080] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and may be is useful infinding intron/exon junctions.

[0081] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-translated regulatory regions.

[0082] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

[0083] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode HRAB, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of HRAB in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressHRAB.

[0084] As will be understood by those of skill in the art, it may beadvantageous to produce HRAB-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0085] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterthe HRAB coding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequence. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, to change codon preference, to produce splicevariants, or other mutations, and so forth.

[0086] In another embodiment of the invention, the natural, modified, orrecombinant polynucleotides encoding HRAB may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HRAB activity, it may be useful toencode a chimeric HRAB protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between a HRAB encoding sequence and theheterologous protein sequence, so that HRAB may be cleaved and purifiedaway from the heterologous moiety.

[0087] In another embodiment, the coding sequences of HRAB may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the HRAB amino acid sequence, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431Apeptide synthesizer (Perkin Elmer).

[0088] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W. H. Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of HRAB, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0089] In order to express a biologically active HRAB, the nucleotidesequence encoding HRAB or functional equivalents, may be inserted intoan appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0090] Methods which are well known to those skilled in the art may beused to construct expression vectors containing a HRAB coding sequenceand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0091] A variety of expression vector/host systems may be utilized tocontain and express a HRAB coding sequences. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0092] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and ptrp-lac hybrids, andthe like may be used. The baculovirus polyhedrin promoter may be used ininsect cells. Promoters or enhancers derived from the genomes of plantcells (e.g., heat shock, RUBISCO; and storage protein genes) or fromplant viruses (e.g., viral promoters or leader sequences) may be clonedinto the vector. In mammalian cell systems, promoters from mammaliangenes or from mammalian viruses are preferable. If it is necessary togenerate a cell line that contains multiple copies of the sequenceencoding HRAB, vectors based on SV40 or EBV may be used with anappropriate selectable marker.

[0093] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for HRAB. For example, whenlarge quantities of HRAB are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding HRAB may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0094] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0095] In cases where plant expression vectors are used, the expressionof a sequence encoding HRAB may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu et al. (1987) EMBO J. 6:307-311;Brisson et al. (1984) Nature 310:511-514). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi et al. (1984) EMBO J. 3:1671-1680; Broglie et al.(1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. CellDiffer. 17:85-105). These constructs can be introduced into plant cellsby direct DNA transformation or pathogen-mediated transfection. Suchtechniques are described in a number of generally available reviews(see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook ofScience and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196;or Weissbach and Weissbach (1988) Methods for Plant Molecular Biology,Academic Press, New York, N.Y., pp. 421-463).

[0096] An insect system may also be used to express HRAB. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequence encoding HRABmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of HRAB will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which HRAB may be expressed (Smith et al. (1983)J. Virol 46:584; Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci.91:3224-3227).

[0097] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, a sequence encoding HRAB may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing HRAB in infected host cells (Logan andShenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0098] Specific initiation signals may also be used to achieve moreefficient translation of a sequence encoding HRAB. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding HRAB, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf D et al. (1994) Results Probl. Cell Differ.20:125-162; Bittner et al. (1987) Methods Enzymol. 153:516-544).

[0099] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0100] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress HRAB may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or separate vector.Following the introduction of the vector, cells may be allowed to growfor 1-2 days in an enriched media before they are switched to selectivemedia. The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

[0101] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr, which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14), and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0102] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequences encoding HRAB isinserted within a marker gene sequence, recombinant cells containingsequences encoding HRAB can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding HRAB under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0103] Alternatively, host cells which contain the coding sequences forHRAB and express HRAB may be identified by a variety of procedures knownto those of skill in the art. These procedures include, but are notlimited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay orimmunoassay techniques which include membrane, solution, or chip basedtechnologies for the detection and/or quantification of the nucleic acidor protein.

[0104] The presence of the polynucleotide sequences encoding HRAB can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding HRAB.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the HRAB-encoding sequence todetect transformants containing DNA or RNA encoding HRAB. As used herein“oligonucleotides” or “oligomers” refer to a nucleic acid sequence of atleast about 10 nucleotides and as many as about 60 nucleotides,preferably about 15 to 30 nucleotides, and more preferably about 20-25nucleotides, which can be used as a probe or amplimer.

[0105] A variety of protocols for detecting and measuring the expressionof HRAB, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson HRAB is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton etal. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul,Minn.) and Maddox et al. (1983) J. Exp. Med. 158:1211-1216).

[0106] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding HRABinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequenceencoding HRAB, or any portion of it, may be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn (Kalamazoo,Mich.); Promega (Madison, Wis.); and U.S. Biochemical Corp. (Cleveland,Ohio). Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

[0107] Host cells transformed with nucleotide sequences encoding HRABmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode HRAB may be designed to contain signal sequences which directsecretion of HRAB through a prokaryotic or eukaryotic cell membrane.Other recombinant constructions may be used to join sequences encodingHRAB to nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and HRAB may be used to facilitate purification. Onesuch expression vector provides for expression of a fusion proteincontaining HRAB] and a nucleic acid encoding 6 histidine residuesfollowed by thioredoxin and an enterokinase cleavage site. The histidineresidues facilitate purification on IMAC (immobilized metal ion affinitychromatography) as described in Porath J et al. (1992, Prot. Exp. Purif.3: 263-281) while the enterokinase cleavage site provides a means forpurifying HRAB from the fusion protein. A discussion of vectors whichcontain fusion proteins is provided in Kroll, D. J. et al. (1993; DNACell Biol. 12:441-453).

[0108] In addition to recombinant production, fragments of HRAB may beproduced by direct peptide synthesis using solid-phase techniques (cfStewart et al. (1969) Solid-Phase Peptide Synthesis, W. H. Freeman Co.,San Francisco, Calif.; Merrifield J. (1963) J. Am. Chem. Soc.85:2149-2154). In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using an Applied Biosystems 431A peptide synthesizer (PerkinElmer). Various fragments of HRAB may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule

[0109] Therapeutics

[0110] In another embodiment of the invention, HRAB or fragments thereofmay be used for therapeutic purposes.

[0111] Chemical and structural homology exists among HRAB proteins,canine rab22 (GI 437987; SEQ ID NO:9), rat Rab-related GTP-bindingprotein (GI 206543; SEQ ID NO:10), mouse Rab17 (GI 297157; SEQ IDNO:11), and mouse Rab6/Rab5-associated protein (GI 722667; SEQ IDNO:12).

[0112] From the homology information provided above, it appears thatHRAB play a role in intracellular transport. Accordingly, in anotherembodiment of the invention, HRAB or derivatives thereof, may be used totreat choroideremia, AIDS, and cancer.

[0113] The antibodies may be generated using methods that are well knownin the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0114] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith HRAB or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0115] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to HRAB have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of HRAB amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0116] Monoclonal antibodies to HRAB may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Koehler et al. (1975) Nature 256:495-497;Kosbor et al. (1983) Immunol. Today 4:72; Cote et al. (1983) Proc. Natl.Acad. Sci. 80:2026-2030; Cole et al. (1985) Monoclonal Antibodies andCancer Therapy, Alan R. Liss Inc., New York, N.Y., pp. 77-96).

[0117] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al. (1984) Proc. Natl.Acad. Sci. 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608;Takeda et al. (1985) Nature 314:452-454). Alternatively, techniquesdescribed for the production of single chain antibodies may be adapted,using methods known in the art, to produce HRAB-specific single chainantibodies. Antibodies with related specificity, but of distinctidiotypic composition, may be generated by chain shuffling from randomcombinatorial immunoglobulin libraries (Kang, A. S. et al. (1991) Proc.Natl. Acad. Sci. 88:11120-3).

[0118] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0119] Antibody fragments which contain specific binding sites for HRABmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse et al. (1989) Science 256:1275-1281).

[0120] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between HRAB and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HRAB epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0121] In another embodiment of the invention, the polynucleotidesencoding HRAB, or any fragment thereof, or antisense sequences, may beused for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding HRAB may be used in situations in which it wouldbe desirable to block the synthesis of the protein. In particular, cellsmay be transformed with sequences complementary to polynucleotidesencoding HRAB. Thus, antisense sequences may be used to modulate HRABactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding HRAB.

[0122] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensepolynucleotides of the gene encoding HRAB. These techniques aredescribed both in Sambrook et al. (supra) and in Ausubel et al. (supra).

[0123] Genes encoding HRAB can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes HRAB. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until all copies aredisabled by endogenous nucleases. Transient expression may last for amonth or more with a non-replicating vector and even longer ifappropriate replication elements are part of the vector system.

[0124] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding HRAB, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the ATG startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0125] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding HRAB.

[0126] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0127] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of RNAmolecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding HRAB. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

[0128] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0129] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro. and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0130] Any of the therapeutic methods described above may be applied toany suitable subject including, for example, mammals such as dogs, cats,cows, horses, rabbits, monkeys, and most preferably, humans.

[0131] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of HRAB,antibodies to HRAB, mimetics, agonists, antagonists, or inhibitors ofHRAB. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0132] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0133] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(MaackPublishing Co., Easton, Pa.).

[0134] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0135] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0136] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0137] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0138] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0139] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0140] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0141] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0142] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of HRAB, such labeling wouldinclude amount, frequency, and method of administration.

[0143] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0144] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0145] A therapeutically effective dose refers to that amount of activeingredient, for example HRAB or fragments thereof, antibodies of HRAB,agonists, antagonists or inhibitors of HRAB, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the ratio LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0146] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0147] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0148] Diagnostics

[0149] In another embodiment, antibodies which specifically bind HRABmay be used for the diagnosis of conditions or diseases characterized byexpression of HRAB, or in assays to monitor patients being treated withHRAB, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for HRAB includemethods which utilize the antibody and a label to detect HRAB in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0150] A variety of protocols including ELISA, RIA, and FACS formeasuring HRAB are known in the art and provide a basis for diagnosingaltered or abnormal levels of HRAB expression. Normal or standard valuesfor HRAB expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to HRAB under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, preferably by photometric, means. Quantities of HRAB expressedin subject, control and disease, samples from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0151] In another embodiment of the invention, the polynucleotidesencoding HRAB may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHRAB may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of HRAB,and to monitor regulation of HRAB levels during therapeuticintervention.

[0152] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HRAB or closely related molecules, may be used to identifynucleic acid sequences which encode HRAB. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding HRAB, alleles, or related sequences.

[0153] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the HRAB encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2, 4, 6, or 8 or from genomic sequence including promoter,enhancer elements, and introns of the naturally occurring HRAB.

[0154] Means for producing specific hybridization probes for DNAsencoding HRAB include the cloning of nucleic acid sequences encodingHRAB or HRAB derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0155] Polynucleotide sequences encoding HRAB may be used for thediagnosis of choroideremia, AIDS, and cancer. The polynucleotidesequences encoding HRAB may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; orin dip stick, pin, ELISA or chip assays utilizing fluids or tissues frompatient biopsies to detect altered HRAB expression. Such qualitative orquantitative methods are well known in the art.

[0156] In a particular aspect, the nucleotide sequences encoding HRABmay be useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequenceencoding HRAB may be labeled by standard methods, and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequence has hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding HRAB in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0157] In order to provide a basis for the diagnosis of diseaseassociated with expression of HRAB, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes HRAB, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0158] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0159] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0160] Additional diagnostic uses for oligonucleotides encoding HRAB mayinvolve the use of PCR. Such oligomers may be chemically synthesized,generated enzymatically, or produced from a recombinant source.Oligomers will preferably consist of two nucleotide sequences, one withsense orientation (5′→3′) and another with antisense (3′←5′), employedunder optimized conditions for identification of a specific gene orcondition. The same two oligomers, nested sets of oligomers, or even adegenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

[0161] Methods which may also be used to quantitate the expression ofHRAB include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0162] In another embodiment of the invention, the nucleic acid sequencewhich encodes HRAB may also be used to generate hybridization probeswhich are useful for mapping the naturally occurring genomic sequence.The sequence may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. Such techniquesinclude FISH, FACS, or artificial chromosome constructions, such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0163] FISH (as described in Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981f). Correlation between the location of thegene encoding HRAB on a physical chromosomal map and a specific disease,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

[0164] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti et al.(1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

[0165] In another embodiment of the invention, HRAB, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenHRAB and the agent being tested, may be measured.

[0166] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to HRAB large numbersof different small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with HRAB, or fragments thereof, and washed. Bound HRAB is thendetected by methods well known in the art. Purified HRAB can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0167] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding HRABspecifically compete with a test compound for binding HRAB. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with HRAB.

[0168] In additional embodiments, the nucleotide sequences which encodeHRAB may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0169] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0170] I Construction of cDNA Libraries

[0171] SYNORAB01 and SYNORAT01

[0172] The cDNA libraries for SYNORAB01 and SYNORAT01 were constructedfrom total RNA from the synovium of a rheumatoid elbow. The rheumatoidsynovial tissue was obtained from UC Davis (lot #48) where it had beenremoved from a 51 year old Asian female and frozen. The frozen tissuewas ground in a mortar and pestle and lysed immediately in a buffercontaining guanidinium isothiocyanate. The lysate was extracted twicewith phenol chloroform at pH 8.0 and centrifuged over a CsCl cushionusing an SW28 rotor in an L8-70M ultracentrifuge (Beckman Instruments).The RNA was precipitated using 0.3 M sodium acetate and 2.5 volumes ofethanol and resuspended in water.

[0173] The RNA for SYNORAB01 which was not DNase treated was used in theSUPERSCRIPT plasmid system for cDNA synthesis and plasmid cloning(catalogue #18248-013; Gibco BRL, Gaithersburg Md.) with the recommendedprotocol. cDNAs were fractionated on a SEPHAROSE CL4B column (catalog#275105, Pharmacia, and those cDNAs exceeding 1 kb were ligated intoPSPORT1. The plasmid was transformed into chemically competent DH5α hostcells (Gibco BRL).

[0174] The RNA for SYNORAT01 was DNase treated for 15 min at 37° C.before library construction. First strand cDNA synthesis wasaccomplished using an oligo d(T) primer/linker which also contained anXhoI restriction site. Second strand synthesis was performed using acombination of DNA polymerase I, E. coli ligase and RNase H, followed bythe addition of an EcoRI adaptor to the blunt ended cDNA. The EcoRIadapted, double-stranded cDNA was then digested with XhoI restrictionenzyme and fractionated on SEPHACRYL S400 to obtain sequences whichexceeded 1000 bp in size. The size selected cDNAs were inserted into theLAMBDAZAP vector system (Stratagene); and the vector, which contains theBLUESCRIPT phagemid (Stratagene), was transformed into cells of E. coli,strain XL1-BLUEMRF (Stratagene).

[0175] In SYNORAT01, the plasmid forms of individual cDNA clones wereobtained by the in vivo excision process. Enzymes from both BLUESCRIPTand a cotransformed f1 helper phage nicked the DNA, initiated new DNAsynthesis, and created the smaller, single-stranded circular phagemidDNA molecules which contained the cDNA insert. The phagemid DNA wasreleased, purified, and used to reinfect fresh host cells (SOLR,Stratagene). Presence of the phagemid which carries the gene forβ-lactamase allowed transformed bacteria to grow on medium containingampicillin.

[0176] EOSIHET02

[0177] The eosinophils used for this library were obtained via aphoresisof a 56 year old Caucasian male patient who had been diagnosed withhypereosinophilic syndrome. The cells were washed twice in phosphatebuffered saline and lysed immediately in a buffer containing guanidiniumisothiocyanate. The lysate was extracted twice with phenol chloroformand centrifuged over a CsCl cushion using an SW28 rotor in an L8-70Multracentrifuge (Beckman Instruments). The RNA was precipitated using0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in waterand DNase treated for 15 min at 37° C. The RNA was isolated using theOLIGOTEX kit (QIAGEN Inc, Chatsworth Calif.) and sent to Stratagene forthe construction of a custom cDNA library.

[0178] First strand cDNA synthesis was accomplished using an oligo d(T)primer/linker which also contained an XhoI restriction site. Secondstrand synthesis was performed using a combination of DNA polymerase I,E. coli ligase and RNase H, followed by the addition of an EcoRI adaptorto the blunt ended cDNA. The EcoRI adapted, double-stranded cDNA wasthen digested with XhoI restriction enzyme and fractionated to obtainsequences which exceeded 800 bp in size. The cDNAs were inserted intothe LAMBDAZAP vector system (Stratagene); then the vector which containsthe BLUESCRIPT phagemid (Stratagene) was transformed into E. coli hostcells strain XL1-BLUEMRF (Stratagene).

[0179] The phagemid forms of individual cDNA clones were obtained by thein vivo excision process. Enzymes from both BLUESCRIPT and acotransformed f1 helper phage nicked the DNA, initiated new DNAsynthesis, and created the smaller, single-stranded circular phagemidmolecules which contained the cDNA insert. The phagemid DNA wasreleased, purified, and used to reinfect fresh host cells (SOLR,Stratagene). Presence of the phagemid which carries the gene forβ-lactamase allowed transformed bacteria to grow on medium containingampicillin.

[0180] TESTTUT02

[0181] The TESTTUT02 cDNA library was constructed from testicle tumortissue obtained from a 31-year old Caucasian male by unilateralorchiectomy. The pathology report indicated that tumor was identified atthe spermatic cord region, and rare foci of residual testicle showedintralobular germ cell neoplasia. Initially, the patient presented withbackache. The patient also had a history of tobacco use. The patient wastaking COLACE (ocusate sodium; Roberts Pharmaceutical Corp., Eatontown,N.J.) and antacids at the time of surgery.

[0182] The frozen tissue was homogenized and lysed using a PolytronPT-3000 (Brinkmann Instruments, Westbury, N.J.) in guanidiniumisothiocyanate solution. The lysate was centrifuged over a 5.7 M CsClcushion using an SW28 rotor in an L8-70M ultracentrifuge (BeckmanInstruments) for 18 hours at 25,000 rpm at ambient temperature. The RNAwas extracted with acid phenol pH 4.0, precipitated using 0.4 M sodiumacetate and 2.5 volumes of ethanol, resuspended in RNAse-free water, andDNase treated at 37° C. The RNA extraction was repeated with acid phenolpH 4.0 and precipitated with sodium acetate and ethanol as before. ThemRNA was then isolated using the OLIGOTEX RNA isolation kit (QIAGENInc.) and used to construct the cDNA library.

[0183] The mRNA was handled according to the recommended protocols inthe SUPERSCRIPT plasmid system for cDNA synthesis and plasmid cloning(Catalog #18248-013, Gibco/BRL). The commercial plasmid PSPORT1(Gibco/BRL) was digested with EcoRI restriction enzyme (New EnglandBiolabs, Beverley, Mass.). The overhanging ends of the plasmid werefilled in using Klenow enzyme (New England Biolabs) and2′-deoxynucleotide 5′-triphosphates (dNTPs). The plasmid wasself-ligated and transformed into the bacterial host, E. coli strain JM109. An intermediate plasmid produced by the bacteria failed to digestwith EcoR I confirming the desired loss of the EcoR I restriction site.

[0184] This intermediate plasmid (PSPORT1-ΔRI) was then digested withHind III restriction enzyme (New England Biolabs) and the overhang wasfilled in with Klenow and dNTPs. A 10-mer linker of sequence 5′ . . .CGGAATTCCG . . . 3′ was phosphorylated and ligated onto the blunt ends.The product of the ligation reaction was digested with EcoR I andself-ligated. Following transformation into JM109 host cells, plasmidswere isolated and screened for the digestibility with EcoR I but notwith Hind III. A single colony which met this criteria was designatedpINCY 1. The plasmid produced by this colony was sequenced and found tocontain several copies of the 10-mer linker. These extra linkers did notpresent a problem as they were eliminated when the vector was preparedfor cloning.

[0185] The plasmid was tested for its ability to incorporate cDNAs froma library prepared using Not I and EcoR I restriction enzymes. Severalclones were sequenced and a single clone containing an insert ofapproximately 0.8 kb was selected to prepare a large quantity of theplasmid for library production. After digestion with Not I and EcoR I,the plasmid and the cDNA insert were isolated on an agarose gel and thevector was purified on a QIAQUICK (QIAGEN, Inc.) column for use inlibrary construction.

[0186] cDNAs were fractionated on a SEPHAROSE CL4B column (Catalog#275105-01, Pharmacia), and those cDNAs exceeding 400 bp were ligatedinto PSPORT1. The plasmid library in PSPORT1 was subsequentlytransformed into DH5α competent cells (Cat. #18258-012, Gibco/BRL).

[0187] II Isolation and Sequencing of cDNA Clones

[0188] SYNORAB01 and SYNORAT01

[0189] Plasmid DNA was purified using the Miniprep kit (Catalogue#77468, Advanced Genetic Technologies Corporation, Gaithersburg Md.), a96-well block kit with reagents for 960 purifications. The recommendedprotocol included with the kit was employed except for the followingchanges. Each of the 96 wells was filled with only 1 ml of sterileTerrific Broth (Catalog #22711, LIFE TECHNOLOGIES, Gaithersburg Md.)with carbenicillin at 25 mg/L and glycerol at 0.4%. After the wells wereinoculated, the bacteria were cultured for 24 hours and lysed with 60 μlof lysis buffer. A centrifugation step (Beckman GS-6R @2900 rpm for 5min; Beckman Instruments) was performed before the contents of the blockwere added to the primary filter plate. The optional step of addingisopropanol to TRIS buffer was not routinely performed. After the laststep in the protocol, samples were transferred to a Beckman 96-wellblock for storage.

[0190] EOSIHET02

[0191] Plasmid DNA was released from the cells and purified using theMiniprep kit (Catalogue #77468; Advanced Genetic TechnologiesCorporation, Gaithersburg Md.) as described above.

[0192] Alternative methods of purifying plasmid DNA include the use ofMAGIC MINIPREPS DNA purification system (Catalogue #A7100, Promega,Madison Wis.) or QIAWELL-8 plasmid, QIAWELL PLUS DNA, and QIAWELL ULTRADNA purification systems (QIAGEN).

[0193] TESTTUT02

[0194] Plasmid DNA was released from the cells and purified using theR.E.A.L PREP 96 plasmid kit for rapid extraction alkaline lysis plasmidminipreps (Catalog #26173, QIAGEN). This kit enabled the simultaneouspurification of 96 samples in a 96-well block using multi-channelreagent dispensers. The recommended protocol was employed except for thefollowing changes: 1) the bacteria were cultured in 1 ml of sterileTerrific Broth (Catalog #22711, LIFE TECHNOLOGIES) with carbenicillin at25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

[0195] The cDNAs were sequenced by the method of Sanger et al. (1975, J.Mol. Biol. 94:441f), using a MICROLAB 2200 (Hamilton, Reno, Nev.) incombination with Peltier thermal cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA sequencing systems; andthe reading frame was determined.

[0196] Most of the sequences disclosed herein were sequenced accordingto standard ABI protocols, using ABI kits (Cat. Nos. 79345, 79339,79340, 79357, 79355). The solution volumes were used at0.25×-1.0×concentrations. Some of the sequences disclosed herein weresequenced using different solutions and dyes which, unless otherwisenoted, came from Amersham Life Science (Cleveland, Ohio).

[0197] First, stock solutions were prepared with HPLC water. Thefollowing solutions were each mixed by vortexing for 2 min: 1) Tris-EDTA(TE) Buffer was prepared by adding 49 ml water to 1 ml 50×Tris-EDTAconcentrate, and 2) 10% Reaction Buffer was prepared by adding 45 mlwater to 5 ml Concentrated Thermo Sequenase (TS) Reaction Buffer.

[0198] Second, 0.2 μM energy transfer (ET) primers were prepared in thefollowing manner. Each primer tube was centrifuged prior to opening toassure that all primer powder was on the bottom of the tube. After eachsolubilization step, the mixture was vortexed for 2 min and thencentrifuged for about 10 sec in a table-top centrifuge. 1 ml of 1×TE wasadded to each primer powder; adenine and cytosine dissolved primers(5-carboxyrhodamine-6G (R6G) and 6-carboxyfluorescein (FAM),respectively), were diluted with 9 ml 1×TE. Guanine and thymine dyes(N,N,N′,N″-tetramethyl-6-carboxyrhodamine (TAM) and6-carboxy-X-rhodamine (ROX), respectively) were diluted with 19 ml 1×TE.

[0199] Next, the sequencing reaction ready mix was prepared asfollows: 1) nucleotides A and C (8 ml of each) were added to 6 ml ETprimer and 18 ml TS reaction buffer; and 2) nucleotides G and T (8 ml ofeach) were added to 6 ml ET primer and 18 ml TS reaction buffer.

[0200] After vortexing for 2 min and centrifuging for 20 sec, theresulting solution was divided into tubes in volumes of 8 ml per tube inorder to make 1×(A,C) and 2×(G,T) solutions.

[0201] After thermal cycling, the A, C, G, and T reactions with each DNAtemplate were combined. Then, 50 μL 100% ethanol was added and thesolution was spun at 4° C. for 30 min. The supernatant was decanted andthe pellet was rinsed with 100 μL 70% ethanol. After being spun for 15min the supernatant was discarded and the pellet was dried for 15 minunder vacuum. The DNA sample was dissolved in 3 μL of formamide/50 mMEDTA. The resulting samples were loaded on wells in volumes of 2 μL perwell for sequencing in ABI sequencers.

[0202] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0203] Each cDNA was compared to sequences in GenBank using a searchalgorithm developed by Applied Biosystems and incorporated into theINHERIT 670 sequence analysis system. In this algorithm, PatternSpecification Language (TRW Inc, Los Angeles, Calif.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments were used to display theresults of the homology search.

[0204] Peptide and protein sequence homologies were ascertained usingthe INHERIT 670 sequence analysis system using the methods similar tothose used in DNA sequence homologies. Pattern Specification Languageand parameter windows were used to search protein databases forsequences containing regions of homology which were scored with aninitial value. Dot-matrix homology plots were examined to distinguishregions of significant homology from chance matches.

[0205] BLAST, which stands for Basic Local Alignment Search Tool(Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990)J. Mol. Biol. 215:403-410), was used to search for local sequencealignments. BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. BLAST is useful for matches which donot contain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

[0206] An HSP consists of two sequence fragments of arbitrary but equallengths whose alignment is locally maximal and for which the alignmentscore meets or exceeds a threshold or cutoff score set by the user. TheBLAST approach is to look for HSPs between a query sequence and adatabase sequence, to evaluate the statistical significance of anymatches found, and to report only those matches which satisfy theuser-selected threshold of significance. The parameter E establishes thestatistically significant threshold for reporting database sequencematches. E is interpreted as the upper bound of the expected frequencyof chance occurrence of an HSP (or set of HSPs) within the context ofthe entire database search. Any database sequence whose match satisfiesE is reported in the program output.

[0207] IV Northern Analysis

[0208] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0209] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0210] The basis of the search is the product score which is defined as:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0211] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0212] The results of northern analysis are reported as a list oflibraries in which the transcript encoding HRAB occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0213] V Extension of HRAB-Encoding Polynucleotides to Full Length or toRecover Regulatory Sequences

[0214] Full length HRAB-encoding nucleic acid sequence (SEQ ID NO:2, 4,6, or 8) is used to design oligonucleotide primers for extending apartial nucleotide sequence to full length or for obtaining 5′ or 3′,intron or other control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence “outward” generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 software (National Biosciences),or another appropriate program, to be 22-30 nucleotides in length, tohave a GC content of 50% or more, and to anneal to the target sequenceat temperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

[0215] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0216] By following the instructions for the XL-PCR kit (Perkin Elmer)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier thermal cycler (PTC200; M. J. Research,Watertown, Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13  4° C. (and holding)

[0217] A 5-10, μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK (QIAGEN Inc.). After recovery of theDNA, Klenow enzyme is used to trim single-stranded, nucleotide overhangscreating blunt ends which facilitate religation and cloning.

[0218] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2×Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2×Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

[0219] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

[0220] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0221] VI Labeling and Use of Hybridization Probes

[0222] Hybridization probes derived from SEQ ID NO:2, 4, 6, or 8 areemployed to screen cDNAs, genomic DNAs, or mRNAs. Although the labelingof oligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences), labeled by combining50 pmol of each oligomer and 250 mCi of [γ-³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). Thelabeled oligonucleotides are substantially purified with SEPHADEX G-25superfine resin column (Pharmacia & Upjohn). A portion containing 10⁷counts per minute of each of the sense and antisense oligonucleotides isused in a typical membrane based hybridization analysis of human genomicDNA digested with one of the following endonucleases (Ase I, Bgl II, EcoRI, Pst I, Xba 1, or Pvu II; DuPont NEN).

[0223] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots, or the blots areexposed in a PhosphorImager cassette (Molecular Dynamics, Sunnyvale,Calif.), hybridization patterns are compared visually.

[0224] VII Antisense Molecules

[0225] Antisense molecules to the HRAB-encoding sequence, or any partthereof, is used to inhibit in vivo or in vitro expression of naturallyoccurring HRAB. Although use of antisense oligonucleotides, comprisingabout 20 base-pairs, is specifically described, essentially the sameprocedure is used with larger cDNA fragments. An oligonucleotide basedon the coding sequences of HRAB, as shown in FIGS. 1A, 1B, and 1C, isused to inhibit expression of naturally occurring HRAB. Thecomplementary oligonucleotide is designed from the most unique 5′sequence as shown in FIGS. 1A, 1B, and 1C and used either to inhibittranscription by preventing promoter binding to the upstreamnontranslated sequence or translation of an HRAB-encoding transcript bypreventing the ribosome from binding. Using an appropriate portion ofthe signal and 5′ sequence of SEQ ID NO:2, 4, 6, or 8, an effectiveantisense oligonucleotide includes any 15-20 nucleotides spanning theregion which translates into the signal or 5′ coding sequence of thepolypeptide as shown in FIGS. 1A, 1B, and 1C.

[0226] VIII Expression of HRAB

[0227] Expression of HRAB is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, PSPORT, previously used for thegeneration of the cDNA library is used to express HRAB in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0228] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of HRAB into the bacterial growth media which can be useddirectly in the following assay for activity.

[0229] IX Demonstration of HRAB Activity

[0230] HRAB's GTP binding activity can be assayed by a techniquedescribed by Brauers A et al (1996, Eur J Biochem 237: 833-840). Samplesof 10 μg HRAB are incubated with tracer ³⁵S guanosine 5′-O-[gamma-thio]triphosphate ([³⁵S] GTP[S]; 300,000 cpm/sample) in a buffer containing20 mM MgCl₂, 1 mM dithiothreitol and 0.1% Triton X-100 in a total volumeof 100 ml. Unlabeled GTP[S] is added and the binding is allowed toproceed at 30° C. for 1 hour. The reaction is terminated by addition of1 ml ice-cold buffer containing 20 mM Tris, pH 8.0, 100 mM NaCl and 25mM MgCl₂. The samples are filtered through nitrocellulose membranes andwashed four times with 1 ml buffer. Samples are placed in scintillationcocktail and radioactivity is measured by scintillation counting.

[0231] X Production of HRAB Specific Antibodies

[0232] HRAB that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2, 4, 6, or 8 is analyzed usingDNASTAR software (DNASTAR Inc) to determine regions of highimmunogenicity and a corresponding oligopolypeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Selection of appropriate epitopes, such as those near the C-terminus orin hydrophilic regions, is described by Ausubel et al. (supra), andothers.

[0233] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems peptide synthesizer Model 431Ausing fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

[0234] XI Purification of Naturally Occurring HRAB Using SpecificAntibodies

[0235] Naturally occurring or recombinant HRAB is substantially purifiedby immunoaffinity chromatography using antibodies specific for HRAB. Animmunoaffinity column is constructed by covalently coupling HRABantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0236] Media containing HRAB is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of HRAB (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HRAB binding (e.g., a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and HRAB is collected.

[0237] XII Identification of Molecules Which Interact with HRAB

[0238] HRAB or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled HRAB, washed and any wells withlabeled HRAB complex are assayed. Data obtained using differentconcentrations of HRAB are used to calculate values for the number,affinity, and association of HRAB with the candidate molecules.

[0239] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 12 184 amino acids amino acid single linear peptide <Unknown>Consensus 1 Met Glu Asp Thr Gly Val Gly Lys Ser Ser Ile Val Cys Arg PheVal 1 5 10 15 Gln Asp His Phe Asp His Asn Ile Ser Pro Thr Ile Gly AlaSer Phe 20 25 30 Met Thr Lys Thr Val Pro Cys Gly Asn Glu Leu His Lys PheLeu Ile 35 40 45 Trp Asp Thr Ala Gly Gln Glu Arg Phe His Ser Leu Ala ProMet Tyr 50 55 60 Tyr Arg Gly Ser Ala Ala Ala Val Ile Val Tyr Asp Ile ThrLys Gln 65 70 75 80 Asp Ser Phe Tyr Thr Leu Lys Lys Trp Val Lys Glu LeuLys Glu His 85 90 95 Gly Pro Glu Asn Ile Val Met Ala Ile Ala Gly Asn LysCys Asp Leu 100 105 110 Ser Asp Ile Arg Glu Val Pro Leu Lys Asp Ala LysGlu Tyr Ala Glu 115 120 125 Ser Ile Gly Ala Ile Val Val Glu Thr Ser AlaLys Asn Ala Ile Asn 130 135 140 Ile Glu Glu Leu Phe Gln Gly Ile Ser ArgGln Ile Pro Pro Leu Asp 145 150 155 160 Pro His Glu Asn Gly Asn Asn GlyThr Ile Lys Val Glu Lys Pro Thr 165 170 175 Met Gln Ala Ser Arg Arg CysCys 180 848 base pairs nucleic acid single linear cDNA <Unknown>Consensus 2 CGCTTTGNTC CGTTTANCCC GGTTCAGANG NGCCGCTGAG CTCCGGCACTGCCTGGCTGC 60 GAGCACATGA TGGCGATACG GGAGCTCAAA GTGTGCCTTC TCGGGGGACTGATGGAACCG 120 ATCTGTTCCC TTACGAAGTG TCACAGTATT GGCAGGACTC TGGACAAGGACAAGGAAGGC 180 TGCATTCCTG TGGCACCACC AGGTGGAAGA TGGAGGACAC TGGGGTTGGGAAATCAAGCA 240 TCGTGTGTCG ATTTGTCCAG GATCACTTTG ACCACAACAT CAGCCCTACTATTGGGGCAT 300 CTTTTATGAC CAAAACTGTG CCTTGTGGAA ATGAACTTCA CAAGTTCCTCATCTGGGACA 360 CTGCTGGTCA GGAACGGTTT CATTCATTGG CTCCCATGTA CTATCGAGGCTCAGCTGCAG 420 CTGTTATCGT GTATGATATT ACCAAGCAGG ATTCATTTTA TACCTTGAAGAAATGGGTCA 480 AGGAGCTGAA AGAACATGGT CCAGAAAACA TTGTAATGGC CATCGCTGGAAACAAGTGCG 540 ACCTCTCAGA TATTAGGGAG GTTCCCCTGA AGGATGCTAA GGAATACGCTGAATCCATAG 600 GTGCCATCGT GGTTGAGACA AGTGCAAAAA ATGCTATTAA TATCGAAGAGCTCTTTCAAG 660 GAATCAGCCG CCAGATCCCA CCCTTGGACC CCCATGAAAA TGGAAACAATGGAACAATCA 720 AAGTTGAGAA GCCAACCATG CAAGCCAGCC GCCGGTGCTG TTGACCCAAGGCCCGTGGTC 780 CACGGTACTT GAAGAAGCCA GAGCCCACAT CCTGTGCACT GCTGAAGGACCCTACNGCTC 840 GGTGGCCT 848 209 amino acids amino acid single linearpeptide <Unknown> Consensus 3 Met Gln Ala Pro His Lys Glu His Leu TyrLys Leu Leu Val Ile Gly 1 5 10 15 Asp Leu Gly Val Gly Lys Thr Ser IleIle Lys Arg Tyr Val His Gln 20 25 30 Leu Phe Ser Gln His Tyr Arg Ala ThrIle Gly Val Asp Phe Ala Leu 35 40 45 Lys Val Leu Asn Trp Asp Ser Arg ThrLeu Val Arg Leu Gln Leu Trp 50 55 60 Asp Ile Ala Gly Gln Glu Arg Phe GlyAsn Met Thr Arg Val Tyr Tyr 65 70 75 80 Lys Glu Ala Val Gly Ala Phe ValVal Phe Asp Ile Ser Arg Ser Ser 85 90 95 Thr Phe Glu Ala Val Leu Lys TrpLys Ser Asp Leu Asp Ser Lys Val 100 105 110 His Leu Pro Asn Gly Ser ProIle Pro Ala Val Leu Leu Ala Asn Lys 115 120 125 Cys Asp Gln Asn Lys AspSer Ser Gln Ser Pro Ser Gln Val Asp Gln 130 135 140 Phe Cys Lys Glu HisGly Phe Ala Gly Trp Phe Glu Thr Ser Ala Lys 145 150 155 160 Asp Asn IleAsn Ile Glu Glu Ala Ala Arg Phe Leu Val Glu Lys Ile 165 170 175 Leu ValAsn His Gln Ser Phe Pro Asn Glu Glu Asn Asp Val Asp Lys 180 185 190 IleLys Leu Asp Gln Glu Thr Leu Arg Ala Glu Asn Lys Ser Gln Cys 195 200 205Cys 890 base pairs nucleic acid single linear cDNA <Unknown> Consensus 4GGCTGCGCTT CCCTGGTCAG GCACGGCACG TCTGGCCGGC CGCCAGGATG CAGGCCCCGC 60ACAAGGAGCA CCTGTACAAG TTGCTGGTGA TTGGCGACCT GGGCGTGGGS AAGACCAGYA 120TCATCAAGCG CTACGTCCAC CAGCTCTTCT CCCAGCACTA CCGGGCCACC ATCGGGGTGG 180ACTTCGCCCT CAAGGTCCTC AACTGGGACA GCAGGACTCT GGTGCGCCTG CAGCTGTGGG 240ACATCGCGGG GCAGGAGCGA TTTGGCAACA TGACCCGAGT ATACTACAAG GAAGCTGTTG 300GTGCTTTTGT AGTCTTTGAT ATATCAAGAA GTTCCACATT TGAGGCAGTC TTAAAATGGA 360AAAGTGATCT GGATAGTAAA GTTCATCTTC CAAATGGCAG CCCTATCCCT GCTGTCCTCT 420TGGCTAACAA ATGTGACCAG AACAAGGACA GTAGCCAGAG TCCTTCCCAG GTGGACCAAT 480TCTGCAAAGA ACATGGCTTT GCCGGATGGT TTGAAACCTC TGCAAAGGAT AACATAAACA 540TAGAGGAAGC TGCCCGGTTC CTAGTGGAGA AGATTCTTGT AAACCACCAA AGCTTTCCTA 600ATGAAGAAAA CGATGTGGAC AAAATTAAGC TAGATCAAGA GACCTTGAGA GCAGAGAACA 660AATCCCAGTG TTGCTGATAT ATGGCTTCTG CTTCTCTTGT GTGTGCCTCA GCTCTGAAGA 720AGTTCCTGAG AATGGGTTAC AGATGTCATG TNAGCTGGGA GTCTTCCNAC ATGTGGNACT 780TCAAAAGGCA GCACNACTGG GCGCNTGCAC TTATTTGAAA ATGGAACTTT GGGAGAAGTA 840TCCCTGCTAN TGGCTCTGTA ACTTAACAGA TGACAATTAG GCTTTTGTNA 890 190 aminoacids amino acid single linear peptide NO <Unknown> Consensus 5 Met AlaGln Ala His Arg Thr Pro Gln Pro Arg Ala Ala Pro Ser Gln 1 5 10 15 ProArg Val Phe Lys Leu Val Leu Leu Gly Ser Gly Ser Val Gly Ala 20 25 30 PhePhe Thr Lys Glu Val Asp Val Gly Ala Thr Ser Leu Lys Leu Glu 35 40 45 IleTrp Asp Thr Ala Gly Gln Glu Lys Tyr His Ser Val Cys His Leu 50 55 60 TyrPhe Arg Gly Ala Asn Ala Ala Leu Leu Val Tyr Asp Ile Thr Arg 65 70 75 80Lys Asp Ser Phe Leu Lys Ala Gln Gln Trp Leu Lys Asp Leu Glu Glu 85 90 95Glu Leu His Pro Gly Glu Val Leu Val Met Leu Val Gly Asn Lys Thr 100 105110 Asp Leu Ser Gln Glu Arg Glu Val Thr Phe Gln Glu Gly Lys Glu Phe 115120 125 Ala Asp Ser Gln Lys Leu Leu Phe Met Glu Thr Ser Ala Lys Leu Asn130 135 140 His Gln Val Ser Glu Val Phe Asn Thr Val Ala Gln Glu Leu LeuGln 145 150 155 160 Arg Ser Asp Glu Glu Gly Gln Ala Leu Arg Gly Asp AlaAla Val Ala 165 170 175 Leu Asn Lys Gly Pro Ala Arg Gln Ala Lys Cys CysAla His 180 185 190 820 base pairs nucleic acid single linear cDNA<Unknown> Consensus 6 CTGCCTGCGG AGGGAAGCAA ACCTTCCCCT GGACCAGAGAGAGGAGAAAG CGGAGACAGG 60 TAGCAACGCT GTGGACTGGT GATGACAGGC TCTTCAGCTCCCTGCAAGTG ACCGGGCCTG 120 GGGAACAGGG CATGGCACAG GCACACAGGA CCCCCCAGCCCAGGGCTGCC CCCAGCCAGC 180 CCCGTGTGTT CAAGCTGGTT CTCCTGGGAA GTGGCTCCGTGGGTGCGTTC TTCACAAAGG 240 AGGTGGATGT GGGTGCCACC TCTCTGAAGC TTGAGATCTGGGACACAGCT GGCCAGGAGA 300 AGTACCACAG CGTCTGCCAC CTCTACTTCA GGGGTGCCAACGCTGCGCTT CTGGTGTACG 360 ACATCACCAG GAAGGATTCC TTCCTCAAGG CTCAGCAGTGGCTGAAGGAC CTGGAGGAGG 420 AGCTGCACCC AGGAGAAGTC CTGGTGATGC TGGTGGGCAACAAGACGGAC CTCAGCCAGG 480 AGCGGGAGGT GACCTTCCAG GAAGGGAAGG AGTTTGCCGACAGCCAGAAG TTGCTGTTCA 540 TGGAAACTTC GGCCAAACTG AACCACCAGG TGTCGGAGGTGTTCAATACA GTGGCCCAAG 600 AGCTACTGCA GAGAAGCGAC GAGGAGGGCC AGGCTCTACGGGGGGATGCA GCTGTGGCTC 660 TGAACAAGGG GCCCGCGAGG CAGGCCAAAT GCTGCGCCCACTAGGTGCAG CCACTCCTGG 720 GGGCTGTGGG GAAGACANCC CCTGCCTGGG CCATGGCCAGCTCTAGGTGG ATTCTGATTC 780 ACTGTCAATG CTGGGTTGCT CCCGAGCCCT AGATGTTCCT820 184 amino acids amino acid single linear peptide <Unknown> Consensus7 Met Ala Ala Gln Lys Asp Gln Gln Lys Asp Ala Glu Ala Glu Gly Leu 1 5 1015 Ser Gly Thr Thr Leu Leu Pro Lys Leu Ile Pro Ser Gly Ala Gly Arg 20 2530 Glu Trp Leu Glu Arg Arg Arg Ala Thr Ile Arg Pro Gly Ala Pro Ser 35 4045 Trp Thr Ser Ser Ala Ser His Gly Pro Ala Thr Trp Glu Ser Cys Ala 50 5560 Ser Ala Val Arg Asn Val Glu Tyr Tyr Gln Ser Asn Tyr Val Phe Val 65 7075 80 Phe Leu Gly Leu Ile Leu Tyr Cys Val Val Thr Ser Pro Met Leu Leu 8590 95 Val Ala Leu Ala Val Phe Phe Gly Ala Cys Tyr Ile Leu Tyr Leu Arg100 105 110 Thr Leu Glu Ser Lys Leu Val Leu Phe Gly Arg Glu Val Ser ProAla 115 120 125 His Gln Tyr Ala Leu Ala Gly Gly Ile Ser Phe Pro Phe PheTrp Leu 130 135 140 Ala Gly Ala Gly Ser Ala Val Phe Trp Val Leu Gly AlaThr Leu Val 145 150 155 160 Val Ile Gly Ser His Ala Ala Phe His Gln IleGlu Ala Val Asp Gly 165 170 175 Glu Glu Leu Gln Met Glu Pro Val 180 757base pairs nucleic acid single linear cDNA <Unknown> Consensus 8GGGTACCGGG CTGGTTACAG CAGCTCTACC CCTCACGACG CAAACATGGC AGCGCAGAAG 60GACCAGCAGA AAGATGCCGA GGCGGAAGGG CTGAGCGGCA CGACCCTGCT GCCGAAGCTG 120ATTCCCTCCG GTGCAGGCCG GGAGTGGCTG GAGCGGCGCC GCGCGACCAT CCGCCCTGGA 180GCACCTTCGT GGACCAGCAG CGCTTCTCAC GGCCCCGCAA CCTGGGAGAG CTGTGCCAGC 240GCTGTACGCA ACGTGGAGTA CTACCAGAGC AACTATGTGT TCGTGTTCCT GGGCCTCATC 300CTGTACTGTG TGGTGACGTC CCCTATGTTG CTGGTGGCTC TGGCTGTCTT TTTCGGCGCC 360TGTTACATTC TCTATCTGCG CACCTTGGAG TCCAAGCTTG TGCTCTTTGG CCGAGAGGTG 420AGCCCAGCGC ATCAGTATGC TCTGGCTGGA GGCATCTCCT TCCCCTTCTT CTGGCTGGCT 480GGTGCGGGCT CGGCCGTCTT CTGGGTGCTG GGAGCCACCC TGGTGGTCAT CGGCTCCCAC 540GCTGCCTTCC ACCAGATTGA GGCTGTGGAC GGGGAGGAGC TGCAGATGGA ACCCGTGTGA 600GGTGTCTTCT GGGACCTGCC GGCCTCCCGG GCCAGCTGCC CCACCCCTGC CCATGCCTGT 660CCTGCACGGS TCTGCTGCTC GGGCCCACAG CGCCGTCCCA TCACAAGCCC GGGGAGGGAT 720CCCGCCTTTR AAAATAAAGC TGTTATGGGT GTCATTC 757 194 amino acids amino acidsingle linear peptide GenBank 437987 9 Met Ala Leu Arg Glu Leu Lys ValCys Leu Leu Gly Asp Thr Gly Val 1 5 10 15 Gly Lys Ser Ser Ile Val TrpArg Phe Val Glu Asp Ser Phe Asp Pro 20 25 30 Asn Ile Asn Pro Thr Ile GlyAla Ser Phe Met Thr Lys Thr Val Gln 35 40 45 Tyr Gln Asn Glu Leu His LysPhe Leu Ile Trp Asp Thr Ala Gly Gln 50 55 60 Glu Ala Phe Arg Ala Leu AlaPro Met Tyr Tyr Arg Gly Ser Ala Ala 65 70 75 80 Ala Ile Ile Val Tyr AspIle Thr Lys Glu Glu Thr Phe Ser Thr Leu 85 90 95 Lys Asn Trp Val Lys GluLeu Arg Gln His Gly Pro Pro Asn Ile Val 100 105 110 Val Ala Ile Ala GlyAsn Lys Cys Asp Leu Ile Asp Val Arg Glu Val 115 120 125 Met Glu Arg AspAla Lys Asp Tyr Ala Asp Ser Ile His Ala Ile Phe 130 135 140 Val Glu ThrSer Ala Lys Asn Ala Ile Asn Ile Asn Glu Leu Phe Ile 145 150 155 160 GluIle Ser Arg Arg Ile Pro Ser Ala Asp Ala Asn Pro Pro Ser Gly 165 170 175Gly Lys Gly Phe Lys Leu Arg Arg Gln Pro Ser Glu Pro Gln Arg Ser 180 185190 Cys Cys 211 amino acids amino acid single linear peptide NO GenBank206543 10 Met Gln Thr Pro His Lys Glu His Leu Tyr Lys Leu Leu Val IleGly 1 5 10 15 Asp Leu Gly Val Gly Lys Thr Ser Ile Ile Lys Arg Tyr ValHis Gln 20 25 30 Asn Phe Ser Ser His Tyr Arg Ala Thr Ile Gly Val Asp PheAla Leu 35 40 45 Lys Val Leu His Trp Asp Pro Glu Thr Val Val Arg Leu GlnLeu Trp 50 55 60 Asp Ile Ala Gly Gln Glu Arg Phe Gly Asn Met Thr Arg ValTyr Tyr 65 70 75 80 Arg Glu Ala Met Gly Ala Phe Ile Val Phe Asp Val ThrArg Pro Ala 85 90 95 Thr Phe Glu Ala Val Ala Lys Trp Lys Asn Asp Leu AspSer Lys Leu 100 105 110 Thr Leu Pro Asn Gly Lys Pro Val Ser Val Val LeuLeu Ala Asn Lys 115 120 125 Cys Asp Gln Gly Lys Asp Val Leu Val Asn AsnGly Leu Lys Met Asp 130 135 140 Gln Phe Cys Lys Glu His Gly Phe Val GlyTrp Phe Glu Thr Ser Ala 145 150 155 160 Lys Glu Asn Ile Asn Ile Asp GluAla Ser Arg Cys Leu Val Lys His 165 170 175 Ile Leu Ala Asn Glu Cys AspPhe Ile Glu Ser Ile Glu Pro Asp Ile 180 185 190 Val Lys Pro His Leu ThrSer Pro Lys Val Val Ser Cys Ser Gly Cys 195 200 205 Ala Lys Ser 210 214amino acids amino acid single linear peptide GenBank 297157 11 Met AlaGln Ala Ala Gly Leu Pro Gln Ala Ser Thr Ala Ser Gly Gln 1 5 10 15 ProTyr Val Ser Lys Leu Val Leu Leu Gly Ser Ser Ser Val Gly Lys 20 25 30 ThrSer Leu Ala Leu Arg Tyr Met Lys Gln Asp Phe Ser Asn Val Leu 35 40 45 ProThr Val Gly Cys Ala Phe Phe Thr Lys Val Leu Asp Leu Gly Ser 50 55 60 SerSer Leu Lys Leu Glu Ile Trp Asp Thr Ala Gly Gln Glu Lys Tyr 65 70 75 80Gln Ser Val Cys His Leu Tyr Phe Arg Gly Ala Asn Ala Ala Leu Leu 85 90 95Val Tyr Asp Ile Thr Arg Lys Asp Ser Phe His Lys Ala Gln Gln Trp 100 105110 Leu Glu Asp Leu Glu Lys Glu Phe Gln Pro Gly Glu Val Val Val Met 115120 125 Leu Val Gly Asn Lys Thr Asp Leu Gly Glu Glu Arg Glu Val Thr Phe130 135 140 Gln Glu Gly Lys Glu Phe Ala Glu Ser Lys Ser Leu Leu Phe MetGlu 145 150 155 160 Thr Ser Ala Lys Leu Asn Tyr Gln Val Ser Glu Ile PheAsn Thr Val 165 170 175 Ala Gln Glu Leu Leu Gln Arg Ala Gly Asp Thr GlySer Ser Arg Pro 180 185 190 Gln Glu Gly Glu Ala Val Ala Leu Asn Gln GluPro Pro Ile Arg Gln 195 200 205 Arg Gln Cys Cys Ala Arg 210 102 aminoacids amino acid single linear peptide GenBank 722667 12 His Glu Asp GlnGln Lys Asp Ala Glu Gly Glu Gly Leu Ser Ala Thr 1 5 10 15 Thr Leu LeuPro Lys Leu Ile Pro Ser Gly Ala Gly Arg Glu Trp Leu 20 25 30 Glu Gln AlaPro Gly Asp His Pro Ala Leu Gly His Leu Ser Trp Thr 35 40 45 Ser Asn ValSer Arg Asp Pro Ala Met Trp Glu Ser Phe Ala Ser Ala 50 55 60 Trp Tyr GlyThr Val Glu Tyr Tyr Gln Ser Asn Tyr Val Phe Val Phe 65 70 75 80 Leu GlyLeu Ile Leu Tyr Cys Val Val Thr Ser Pro Met Leu Leu Val 85 90 95 Ala LeuAla Val Phe Phe 100

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, and SEQ ID NO:7, b) a polypeptide comprising a naturally occurringamino acid sequence at least 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, and SEQ ID NO:7, c) a biologically active fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7,and d) an immunogenic fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, and SEQ ID NO:7.
 2. An isolated polypeptide of claim 1having a sequence selected from the group consisting of SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, and SEQ ID NO:7.
 3. An isolated polynucleotideencoding a polypeptide of claim
 1. 4. An isolated polynucleotideencoding a polypeptide of claim
 2. 5. An isolated polynucleotide ofclaim 4, having a sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.
 6. A recombinantpolynucleotide comprising a promoter sequence operably linked to apolynucleotide of claim
 3. 7. A cell transformed with a recombinantpolynucleotide of claim
 6. 8. A transgenic organism comprising arecombinant polynucleotide of claim
 6. 9. A method for producing apolypeptide of claim 1, the method comprising: a) culturing a cell underconditions suitable for expression of the polypeptide, wherein said cellis transformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide encoding the polypeptide of claim 1, and b) recoveringthe polypeptide so expressed.
 10. A method of claim 9, wherein thepolypeptide has an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.11. An isolated antibody which specifically binds to a polypeptide ofclaim
 1. 12. An isolated polynucleotide selected from the groupconsisting of: a) a polynucleotide comprising a polynucleotide sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, and SEQ ID NO:8, b) a polynucleotide comprising a naturallyoccurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, c) a polynucleotidecomplementary to a polynucleotide of a), d) a polynucleotidecomplementary to a polynucleotide of b), and e) an RNA equivalent ofa)-d).
 13. An isolated polynucleotide comprising at least 60 contiguousnucleotides of a polynucleotide of claim
 12. 14. A method for detectinga target polynucleotide in a sample, said target polynucleotide having asequence of a polynucleotide of claim 12, the method comprising: a)hybridizing the sample with a probe comprising at least 20 contiguousnucleotides comprising a sequence complementary to said targetpolynucleotide in the sample, and which probe specifically hybridizes tosaid target polynucleotide, under conditions whereby a hybridizationcomplex is formed between said probe and said target polynucleotide orfragments thereof, and b) detecting the presence or absence of saidhybridization complex, and, optionally, if present, the amount thereof.15. A method of claim 14, wherein the probe comprises at least 60contiguous nucleotides.
 16. A method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide has an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, and SEQ ID NO:7.
 19. A method for treating a disease orcondition associated with decreased expression of functional HRAB,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method for screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional HRAB, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method for screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional HRAB, comprising administering to a patientin need of such treatment a composition of claim
 24. 26. A method ofscreening for a compound that specifically binds to the polypeptide ofclaim 1, said method comprising the steps of: a) combining thepolypeptide of claim 1 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 1 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim
 1. 27. A method of screening for acompound that modulates the activity of the polypeptide of claim 1, saidmethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method for screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a polynucleotide sequence of claim 5, themethod comprising: a) exposing a sample comprising the targetpolynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 29. A method for assessing toxicityof a test compound, said method comprising: a) treating a biologicalsample containing nucleic acids with the test compound; b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 12 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 12 or fragment thereof; c) quantifying theamount of hybridization complex; and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of HRAB in a biological sample comprising the steps of: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex; and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of HRAB in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofHRAB in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 34. 36. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 11comprising: a) immunizing an animal with a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, and SEQ ID NO:7, or an immunogenic fragment thereof,under conditions to elicit an antibody response; b) isolating antibodiesfrom said animal; and c) screening the isolated antibodies with thepolypeptide, thereby identifying a polyclonal antibody which bindsspecifically to a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, andSEQ ID NO:7.
 37. An antibody produced by a method of claim
 36. 38. Acomposition comprising the antibody of claim 37 and a suitable carrier.39. A method of making a monoclonal antibody with the specificity of theantibody of claim 11 comprising: a) immunizing an animal with apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, oran immunogenic fragment thereof, under conditions to elicit an antibodyresponse; b) isolating antibody producing cells from the animal; c)fusing the antibody producing cells with immortalized cells to formmonoclonal antibody-producing hybridoma cells; d) culturing thehybridoma cells; and e) isolating from the culture monoclonal antibodywhich binds specifically to a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, and SEQ ID NO:7.
 40. A monoclonal antibody produced by a method ofclaim
 39. 41. A composition comprising the antibody of claim 40 and asuitable carrier.
 42. The antibody of claim 11, wherein the antibody isproduced by screening a Fab expression library.
 43. The antibody ofclaim 11, wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 44. A method for detecting a polypeptide havingan amino acid sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7 in a sample, comprisingthe steps of: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide; and b) detecting specific binding, wherein specific bindingindicates the presence of a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, and SEQ ID NO:7 in the sample.
 45. A method of purifying apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7from a sample, the method comprising: a) incubating the antibody ofclaim 11 with a sample under conditions to allow specific binding of theantibody and the polypeptide; and b) separating the antibody from thesample and obtaining the purified polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, and SEQ ID NO:7.
 46. A microarray wherein at least oneelement of the microarray is a polynucleotide of claim
 13. 47. A methodfor generating a transcript image of a sample which containspolynucleotides, the method comprising the steps of: a) labeling thepolynucleotides of the sample, b) contacting the elements of themicroarray of claim 46 with the labeled polynucleotides of the sampleunder conditions suitable for the formation of a hybridization complex,and c) quantifying the expression of the polynucleotides in the sample.48. An array comprising different nucleotide molecules affixed indistinct physical locations on a solid substrate, wherein at least oneof said nucleotide molecules comprises a first oligonucleotide orpolynucleotide sequence specifically hybridizable with at least 30contiguous nucleotides of a target polynucleotide, said targetpolynucleotide having a sequence of claim
 12. 49. An array of claim 48,wherein said first oligonucleotide or polynucleotide sequence iscompletely complementary to at least 30 contiguous nucleotides of saidtarget polynucleotide.
 50. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 60 contiguous nucleotides of said target polynucleotide. 51.An array of claim 48, which is a microarray.
 52. An array of claim 48,further comprising said target polynucleotide hybridized to said firstoligonucleotide or polynucleotide.
 53. An array of claim 48, wherein alinker joins at least one of said nucleotide molecules to said solidsubstrate.
 54. An array of claim 48, wherein each distinct physicallocation on the substrate contains multiple nucleotide molecules havingthe same sequence, and each distinct physical location on the substratecontains nucleotide molecules having a sequence which differs from thesequence of nucleotide molecules at another physical location on thesubstrate.
 55. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 56. A polypeptide of claim 1, comprising theamino acid sequence of SEQ ID NO:3.
 57. A polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO:5.
 58. A polypeptide ofclaim 1, comprising the amino acid sequence of SEQ ID NO:7.
 59. Apolynucleotide of claim 12, comprising the polynucleotide sequence ofSEQ ID NO:2.
 60. A polynucleotide of claim 12, comprising thepolynucleotide sequence of SEQ ID NO:4.
 61. A polynucleotide of claim12, comprising the polynucleotide sequence of SEQ ID NO:6.
 62. Apolynucleotide of claim 12, comprising the polynucleotide sequence ofSEQ ID NO:8.